Potentiation of the immune response

ABSTRACT

A method for stimulating proliferation of T-cells containing cytoplasmic post-prolyl dipeptidase activity; the method, in one aspect, involves contacting the T-cells with an organic compound at a concentration below 10 −8 M, wherein the compound is characterized in that: (a) it is capable of crossing the membrane of T-cells to enter the cytoplasm, (b) it binds to the dipeptidase activity at a concentration of below 10 −8 M, and thus (c) stimulates proliferation of the T-cells at that concentration.

RELATED APPLICATIONS

This application is a continuation and claims priority under 35 U.S.C. §120 to application U.S. Ser. No. 09/491,855, filed January 26, entitled,“Potentiation of the Immune Response,” pending, which is a divisionalapplication and claims priority under 35 U.S.C. § 120 to U.S. Ser. No.08/852,395, filed May 7, 1997, entitled “Potentiation of the ImmuneResponse,” now issued as U.S. Pat. No. 6,040,145, the entire contents ofeach of which are incorporated herein by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under NIH grant No.AI36696, and the Government therefore has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

This invention relates to treatment of viral infections using organiccompounds which interact with T-cell enzymes.

One of the classic markers of full-blown AIDS resulting from long-terminfection with HIV-1 is a severe depletion of CD4⁺ T-cells, which are akey component of the immune system. Attempts have been made to increasethe CD4⁺ counts of AIDS patients, and some of these efforts, notablytreatment with protease inhibitors, have met with considerable success.Other approaches, e.g., stimulation of the immune response byvaccination with viral peptides, have been less successful. The reasonsfor CD4⁺ depletion in AIDS, and resistance of CD4⁺ cells to stimulationby some therapies are not fully understood.

SUMMARY OF THE INVENTION

We have discovered that the activation state of human T-cells can beaffected by compounds which interact with a cytoplasmic post-prolyldipeptidase activity which has similarities to, but is distinct from,the membrane-bound T-cell serine protease CD26. The compounds useful inthe invention are inhibitors of this activity, which is, innaturally-occurring T-cells in healthy individuals, involved inprotection of T-cells from apoptosis, or programmed cell death. Thus, inhigh concentrations, the inhibitors hasten the death of T-cells, byinhibiting the protective enzyme. We have discovered, surprisingly, thatat low concentrations the inhibitors exhibit a paradoxical effect: theyare potent stimulators of T-cell activity in HIV-infected individuals.The concentrations of inhibitor which induce this T-cell stimulatoryresponse at very low (on the order of 10⁻⁸-10⁻¹²M), and therefore theinhibitors can be used with minimal side effects, even if, in largerdoses, the inhibitors would be toxic.

Our hypothesis is that the resistance to full activation observed inT-cells of HIV-infected individuals involves a blocking of thecytoplasmic enzymatic activity discussed above. We believe that thisblocking of activation, involving this cytoplasmic activity, preventsdifferentiation of T-cells of HIV-infected individuals into effectorcells, eventually leading to T-cell death.

Thus, the invention features a method for stimulating proliferation ofT-cells of a human patient suffering from a disease state characterizedby the inability of the patients' T-cells to respond normally to T-cellproliferation-inducing stimuli; the method involves contacting theT-cells with an organic compound at a concentration below 10⁻⁸M, whereinthe compound is characterized in that it binds to the post-prolylcleaving dipeptidase activity present in the cytoplasm of human T-cells,e.g., CD4⁺ cells or Jurkat cells.

Treatment according to the invention can be in vitro or in vivo. In invivo therapy, the enzyme-interacting compound of the invention isadministered such that the blood concentration in the patient (e.g., anHIV-infected patient) is below 10⁻¹¹. The compounds can also be used invitro at low concentrations to stimulate proliferation of non-infected,beneficial T-cells, such as CD4⁺ cells and CTL's. In this embodiment,PBMC are isolated from a patient and incubated with a concentration oflower than 10⁻⁸M of the compound, to bring about proliferation ofT-cells, which are then reinfased into the patient.

We believe that administration of low concentrations of the inhibitorsof the invention may have an allosteric effect such that the T-cellcytoplasmic enzyme, which is a multimeric (i.e., multiple subunit)enzyme, exhibits an increased affinity of the enzyme for its naturalsubstrate or ligands, allowing the previously blocked T-cell to proceedto full activation, and hence survival, proliferation, and interleukin-2production. Stimulation of the T-cell immune response in HIV-infectedpatients according to the invention yields increased numbers of immuneeffector cells, which can fight both HP, itself and other opportunisticpathogens.

Treatment according to the invention has the advantages of specificityand low toxicity, not just because of the low concentrations ofinhibitor which can be used, but also because, in T-cells of patientsnot infected with a virus such as HIV, the inhibitors have nodiscernable effect. Furthermore, treatment according to the inventionadvantageously does not necessarily require in vitro manipulation of theT-cells from HIV-infected patients. Furthermore, no immunization isrequired, and treatment will be effective even where HIV proteins havemutated because the therapy targets a cellular enzyme. The fact that, inT-cells treated according to the invention in vitro, no increase in thelevel of the HIV protein p24 is observed, probably indicates that theT-cells which are infected with HIV are not stimulated by the low doseinhibitor treatment of the invention.

The invention also permits immunization of HIV-infected patients with,e.g., HIV peptides. Under normal circumstances, such patients cannot bevaccinated because of the defect in the T-cell stimulation pathway. Useof inhibitors in low doses as adjuvants can render T-cells responsive tovaccination with HIV antigens, in particular peptides.

Treatment of HIV-infected patients with low doses of inhibitorsaccording to the invention can also enhance the activity of other AIDSdrugs, in particular protease inhibitors. We have found that treatmentaccording to the invention generally fails to bring about an increase inCD4⁻ count in patients whose CD4⁻ count is already very low, i.e., belowabout 400. In such patients, the CD4⁻ count can be increased to abovethis level using known protease inhibitors, and the newly generated CD4⁻T-cells resulting from such treatment are particularly susceptible tothe stimulatory effects of treatment according to the invention, leadingto an optimal combination AIDS therapy. Preferably, the drugs areadministered orally.

The low dose administration of inhibitors of the invention can also beused to produce an adjuvant effect in HIV-negative individuals, who areto be immunized with peptides or other viral antigens; this mode ofvaccination can be used for prophylaxis for HIV, as well as any otherviral pathogen. Ordinarily, meaningful cytolytic T-lymphocyte (“CTL”)responses, both in vitro and in vivo, have been difficult to achievewith peptide immunization. The invention should make it possible toproduce significant CTL responses to viral peptides, e.g., peptides frominfluenza, HIV, human papilloma virus, and herpes peptides. Thisadjuvant effect can also be used to stimulate CTL responses to peptideantigens from other pathogens as well, e.g., pathogenic bacteria such astoxigenic E. coli, and protozoan pathogens such as the pathogens whichare the causative agents of malaria and amoebic dysentery. Thecompounds, when used as adjuvants, are preferably administered orally.

The invention provides a new and highly advantageous method ofpotentiating the immune response in both HIV infected and uninfectedpatients, in methods employing extremely low concentrations ofinhibitors which, at these concentrations, exhibit a paradoxical effect(i.e., they act as stimulatory rather than inhibitory molecules, as theywould at higher concentrations). The very low concentrations employedaccording to the invention allows treatment with minimal side reactionsand toxicity. The specificity of the treatment of the invention alsoavoids such adverse effects, which are seen, for example, in treatmentwith immune stimulatory compounds such as interleukin-2.

Other features and advantages of the invention will be apparent from thefollowing detailed description thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pair of graphs showing the lymphocyte stimulatory effect oftreatment of the invention on peripheral blood mononuclear cells (PBMC)from HIV-infected and uninfected patients.

FIG. 2 is a graph illustrating the T-cell stimulatory effects of twoinhibitory compounds used according to the invention.

FIGS. 3, 4, and 9 are graphs showing stimulatory effects of treatmentaccording to the invention in lymphocytes of HIV-infected patients,compared to treatment using two control compounds.

FIG. 5 is a graph illustrating a stimulatory effect of an inhibitoraccording to the invention on PBMC in vitro, showing the correlationwith CD4⁺ counts. The data are plotted as the natural 10 g of thestimulation index (vertical dimension) versus the natural log of theCD4⁻ count of the patient (horizontal dimension).

FIG. 6 is a histogram demonstrating that an inhibitor according to theinvention induces dose-dependent apoptosis in resting T-cells (thesedosages are higher than the extremely low doses used according to theinvention).

FIG. 7 is a histogram demonstrating that an inhibitor according to theinvention induces, at higher doses than in the invention, dose-dependentapoptosis in both CD26⁺ and CD26⁺ populations of PBMC.

FIG. 8 is a graph showing that an inhibitor of CD26 inhibited thecytoplasmic enzyme as well.

DETAILED DESCRIPTION

Therapeutic Compounds

Any organic compound can be used according to the invention whichexhibits the following properties: (1) it is capable of crossing themembrane of human T-cells to reach the cytoplasm, where the compound can(2) intereact with the cytoplasmic dipeptidase present in the T-cells,in order to (3) stimulate activation/proliferation of T-cells (and mostpreferably CD4⁺ cells or CTL's) at concentrations below 10⁻⁸M.

A simple screening method is described below for the identification ofcompounds which are candidate therapeutic compounds according to theinvention.

Substrate and Enzyme Preparation

The first step is to provide a cytoplasmic enzyme preparation. Thepreparation need not be a pure enzyme sample; a crude cytoplasmicextract is sufficient to screw compounds for the desired activity. Theextract can be prepared from any human T-cell line which is negative forCD26; an example of such a suitable cell line is the commerciallyavailable Jurkat cell line.

A suitable enzyme-containing cell extract can be prepared as follows.First, Jurkat cells (10⁶-10¹¹ cells) are grown and a cell pellet isobtained by centrifugation. The cell pellet is stored in frozencondition.

For use in the assay, the frozen pellet is thawed by the addition of icecold lysis buffer, in the amount of approximately 1 ml per 10⁸ cells.The liquified material is homogenized with ten strokes of a Douncehomogenizer, and then clarified by centrifugation at 1500 g. Thesupernatant is removed (and saved), and the 1500 g. pellet isresuspended in lysis buffer and homogenized with ten strokes of a Douncehomogenizer. Clarification is again carried out by centrifugation at1500 g, 4° C.

The 1500 g supernatants are then combined, and EDTA is added to 5 mM.The resultant liquid is centrifuged at 75,000 g at 4° C. for twentyminutes, and the supernatant is then removed and centrifuged at 175,000g. at 4° C., for 60 minutes. The resultant supernatant, containing thecytosolic extract, is the DPPV activity-containing preparation used inthe assay, described below, for candidate therapeutic compounds of theinvention.

The assay is based on our observation that the T-cell cytoplasmic enzymeof interest is a post-prolyl cleaving serine protease. We thereforechose as a reporter substrate a compound which contains proline in thepenultimate position; any of a number of substrates meeting thisrequirement can be used. In the assay described herein, we employed afluorescent cleavage assay using the substrate AlaProAFC. Alternatively,a colorimetric assay can be carried out using as a substrateGly-Pro-pNA. The choice of terminal amino acid is not critical, providedthat the substrate contain a fire: terminal amino group.

In the assay we carried out, we employed a fluorescence spectrometer forexcitation at 400 nm and emission at 505 nm. The spectrometer wascalibrated for fluoresence intensity of 0.000=10 mM HEPES, pH 7.4; andfluoresence intensity of 1.000=10 Mm HEPES, 1 μM AFC.

To carry out the assay, between 10 and 100 μl of enzyme extact, above,is diluted to 1 ml with 10 mM HEPES, pH 7.4, containing 10 mMAla-Pro-AFC. At least one extract/substrate sample is run without testcompound, to provide a standard for comparison with the test sample.

In the test samples, multiple samples are run containing varyingconcentrations, down to 10⁻⁸M, of the test compound. The sample (with orwithout test compound) is placed in a cuvette, and inserted into afluoresent spectrometer. Enzymatic activity is measured as theaccumulation of fluoresence intensity (i.e., substrate cleavage product)over time (1 min.). A compound is identified as an inhibitor if theaccumulative fluoresence is decreased as a result of the presence of theinhibiting compound.

Once a compound has been identified as an enzymatic inhibitor, asdescribed above, further assays are carried out to determine whether thecompound is capable of moving across the T-cell membrane into thecytoplasm; this is an assay which can be carried out using well-knowntechniques.

If desired, additional in vitro assays can be carried out usingcandidate compounds of the invention, prior to their use in vivo. Onesuch assay employs the candidate compound at a very low concentration,in a test designed to determine whether at low concentrations thecompound can stimulate the proliferation of PBMC from HIV infectedpatients in vitro. As is shown in the data of FIG. 4, stimulation can bemeasured by, e.g., incorporation of a labelled nucleotide.

The compounds can also be tested at higher doses to determine whetherthey exhibit the opposite effect of proliferation, as above, i.e.,dose-dependent apoptosis caused by enzyme inhibition, as in theexperiments of FIG. 6.

Candidate Compounds

As is discussed above, compounds which are potentially capable ofapoptosis induction at high doses and proliferation induction at lowdoses are those which, at normal or high doses, inhibit cytoplasmicT-cell dipeptidase, and can cross the T-cell membrane into the T-cellcytoplasm, where the enzyme interaction occurs. The compounds thusshould be organic compounds which have a free amino group at the aminoterminus; a proline or proline analog at the penultimate position; andan enzyme binding site which mimics the post-prolyl cleavage site ofcytoplasmic dipeptidase.

A number of known classes of compounds can be screened and usedaccording to the invention. Once such class are CD26 (i.e., DPPIV)inhibitors, including those described in Bachovchin et al. U.S. Pat. No.4,935,493, hereby incorporated by reference. In the '493 patent, thereare described compounds having the structure:

where each D1 and D2, independently, is a hydroxyl group or a groupwhich is capable of being hydrolyzed to a hydroxyl group in aqueoussolution at physiological pH; and X comprises an amino acid or a peptidewhich mimics the site of the substrate recognized by a post-prolylcleaving enzyme.

The compounds in the '493 patent are inhibitors of CD26, and are alsocandidate inhibitors of the invention. As is discussed above, because ofthe low concentrations of compounds used according to the invention, itis acceptable to use, in the invention, a compound which interacts notonly with the cytoplasmic enzyme, but also CD26.

The class of compounds described in the '493 patent are also discussedand exemplified in Takacs et al. U.S. patent application Ser. No.07/923,337, corresponding to PCT Application No. WO94/03055, herebyincorporated by reference. In this application, one of the families ofmolecules in the '493 patent is described as the “Xaa-boroPromolecules.” exemplified by Ala-boroPro, Pro-boroPro, and Gly-boroPro.These Xaa-boroPro molecules are all candidate compounds for use in themethods of the present invention. Two of these compounds are used insome of the examples described below; those compounds are Lys-boroPro(“KPB”), and Val-boroPro (“VBP”).

EXAMPLE 1

Peripheral blood mononuclear cells (PBMC) were obtained by standardmethods from HIV-infected individuals, and from uninfected individuals.Varying dosages of KBP or VBP were contacted with the PBMC in vitro, andstimulation of proliferation was measured by incorporation of ³Hthymidine (cpm). The results of these experiments are shown in FIG. 1:very low doses of the Val-boroPro and Lys-boroPro stimulatedproliferation of PBMC from HIV-infected patients, but not PBMC fromuninfected patients.

As shown in FIG. 1, at no concentration of the boroPro enzyme inhibitordid it affect the PBMC from uninfected individuals. The inhibitor, atmoderate concentrations, also did not cause proliferation of PBMC fromHIV-infected individuals, but it did cause marked proliferation at verylow concentrations (10⁻⁹ and 10⁻¹⁰). These results are consistent withour hypothesis, discussed above, that, at low concentrations, theseenzyme inhibitors exhibit a paradoxical effect: rather than inhibitingthe apoptosis-controlling cytoplasmic T-cell enzyme, they interact withthat enzyme in a manner which blocks inactivation and causesproliferation of T-cells.

Concordant results are shown in FIG. 2, a histogram showing that lowdoses of Lys-boroPro and Val-boroPro cause proliferation of PBMC ofHIV-infected patients, while higher doses (10⁻⁴M) do not have thiseffect.

The same results are shown in FIGS. 3, 4, 9, and 10 which also presentdata for two control compounds OKT3, and PHA, both of which arenon-specific mitogens.

Referring to FIG. 5, data are presented in a form which shows that lowconcentrations of the inhibitors of the invention have little effect onthe PBMC of HIV-infected patients whose CD4 counts are lower than about400 (the clinical indication for AIDS). In the graph of FIG. 5, thenatural log of the stimulation index (the vertical axis) is plottedagainst the natural log of the CD4 count of the patients; as shown,above a count of 400 there is particularly significant stimulation ofproliferation.

FIG. 6 is a graph demonstrating that purified T-cells are highlysensitive to cytoplasmic T-cell dipeptidase inhibitors in moderateconcentrations. CD19⁻ B cells and CD4⁻/CD8⁻ T-cells were isolated tohigh purity and incubated overnight in Val-boroPro. The amount of celldeath was determined by 7AAD flow cytometry analysis. Data represent %of cell death from duplicate samples. These data are consistent with ourhypothesis that the inhibitors, in moderate concentration, inhibit acytoplasmic enzyme which ordinarily protects against apoptosis.

FIG. 7 presents data demonstrating that CD26⁻ and CD26⁻ PBMC are equallysusceptible to T-cell cytoplasmic enzyme inhibitor-induced death, wherethe inhibitor is administered in moderate concentrations. CD26⁻ andCD26⁻ populations were incubated overnight in the presence or absence ofvarious concentrations of Val-boroPro. The amount of cell death wasdetermined by 7AAD flow cytometry analysis. Data represent mean % ofdeath from duplicate samples. These data indicate thatapoptosis-inhibiting T-cell cytoplasmic enzyme is present in both CD26⁻and CD26⁻ T-cells.

FIG. 8 presents data showing the effects of an inhibitor useful in theinvention, Val-boroPro. The experiments were carried out using twopreparations: purified DPPIV (i.e., CD26), and Jurkat T-cell cytoplasmicextract, described above (Jurkat cells contain the cytoplasmic T-cellenzyme, but do not bear CD26 on their surfaces). These preparations wereincubated with varying concentrations of Val-boroPro, and enzymaticactivity was determined by measuring the accumulation of the fluorescentcleavage product 7-amino-4-trifluoromethylcoumarin (AFC) released fromthe substrate Ala-ProAFC upon enzymatic cleavage. Val-boroPro inhibitedboth the enzyme DPPIV and the cytoplasmic T-cell enzyme in the Jurkatpreparation.

Other embodiments are within the following claims.

1-19. (canceled)
 20. A method for stimulating an antigen-specific immuneresponse in a subject comprising administering to an HIV-negativesubject in need thereof a pathogenic peptide antigen and a compoundhaving the structure

wherein each D₁ and D₂, independently, is a hydroxyl group or a groupwhich is capable of being hydrolyzed to a hydroxyl group in aqueoussolution at physiological pH; and X comprises an amino acid or a peptidewhich mimics the site of the substrate recognized by a post-prolylcleaving enzyme.
 21. The method of claim 20, wherein the pathogenicpeptide antigen is a viral peptide antigen.
 22. The method of claim 21,wherein the viral peptide antigen is derived from HIV, influenza virus,human papilloma virus, and herpes virus.
 23. The method of claim 20,wherein the pathogenic peptide antigen is a bacterial peptide antigen.24. The method of claim 23, wherein the bacterial peptide antigen isderived from E. coli.
 25. The method of claim 20, wherein the pathogenicpeptide antigen is a protozoan peptide antigen.
 26. The method of claim25, wherein the protozoan peptide antigen is derived from a malariacausing agent or an amoebic dysentery causing agent.
 27. The method ofclaim 20, wherein the subject has an infection other than HIV infection.28. The method of claim 20, wherein the subject is at risk of developingan infection.
 29. The method of claim 27, wherein the infection isselected from the group consisting of a bacterial infection, a viralinfection or a protozoan infection.
 30. The method of claim 28, whereinthe infection is selected from the group consisting of a bacterialinfection, a viral infection or a protozoan infection.
 31. The method ofclaim 20, wherein the compound is administered orally.
 32. The method ofclaim 20, wherein the compound is selected from the group consisting ofAla-boroPro, Pro-boroPro, Gly-boroPro and Lys-boroPro.
 33. The method ofclaim 20, wherein the compound is Val-boroPro.
 34. The method of claim20, wherein the antigen-specific immune response is a cytolytic Tlymphocyte response.
 35. The method of claim 20, wherein X is an aminoacid.
 36. The method of claim 20, wherein X is a peptide.